Journal of Plant Physiology 176 (2015) 192–201

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Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph

Review article

Plant glutathione peroxidases: Emerging role of the antioxidant enzymes in plant development and stress responses Krisztina Bela a , Edit Horváth a , Ágnes Gallé a , László Szabados b , Irma Tari a , Jolán Csiszár a,∗ a b

Department of Plant Biology, Faculty of Science and Informatics, University of Szeged, Közép fasor 52., H-6726 Szeged, Hungary Institute of Plant Biology, Biological Research Centre of HAS, Temesvári krt. 62., H-6726 Szeged, Hungary

a r t i c l e

i n f o

Article history: Received 6 October 2014 Received in revised form 15 December 2014 Accepted 15 December 2014 Available online 13 January 2015 Keywords: Antioxidant enzyme Plant glutathione peroxidase Reactive oxygen species Redox regulation

a b s t r a c t The plant glutathione peroxidase (GPX) family consists of multiple isoenzymes with distinct subcellular locations which exhibit different tissue-specific expression patterns and environmental stress responses. Contrary to most of their counterparts in animal cells, plant GPXs contain cysteine instead of selenocysteine in their active site and while some of them have both glutathione peroxidase and thioredoxin peroxidase functions, the thioredoxin regenerating system is much more efficient in vitro than the glutathione system. At present, the function of these enzymes in plants is not completely understood. The occurrence of thiol-dependent activities of plant GPX isoenzymes suggests that – besides detoxification of H2 O2 and organic hydroperoxides – they may be involved in regulation of the cellular redox homeostasis by maintaining the thiol/disulfide or NADPH/NADP+ balance. GPXs may represent a link existing between the glutathione- and the thioredoxin-based system. The various thiol buffers, including Trx, can affect a number of redox reactions in the cells most probably via modulation of thiol status. It is still required to identify the in vivo reductant for particular GPX isoenzymes and partners that GPXs interact with specifically. Recent evidence suggests that plant GPXs does not only protect cells from stress induced oxidative damage but they can be implicated in plant growth and development. Following a more general introduction, this study summarizes present knowledge on plant GPXs, highlighting the results on gene expression analysis, regulation and signaling of Arabidopsis thaliana GPXs and also suggests some perspectives for future research. © 2015 Elsevier GmbH. All rights reserved.

Contents The plant glutathione peroxidases are members of the thioredoxin-dependent peroxidase family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The mammalian glutathione peroxidases (GPxs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The plant glutathione peroxidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The role of plant GPXs in stress responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The plant GPXs and the oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GPXs and the redox regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Arabidopsis thaliana glutathione peroxidases—Lessons from a model plant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AtGPX1 and AtGPX7—The chloroplastic isoenzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AtGPX2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AtGPX3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: ABA, abscisic acid; ABI, abscisic acid insensitive; APX, ascorbate peroxidase; AS, antisense; ASC, ascorbate; CAT, catalase; DHA, dehydroascorbic acid; GA, gibberellic acid; GPX, glutathione peroxidase; GPx4/PHGPX, phospholipid hydroperoxide glutathione peroxidase/animal GPx4 enzime; GR, glutathione reductase; GRX, glutaredoxin; GSH, reduced glutathione; GSSG, oxidized glutathione/glutathione disulfide; GST, glutathione transferase; IAA, indole-3-acetic acid; MeJA, methyl jasmonate; POD, guaiacol peroxidase; Prx, peroxiredoxin/thioredoxin peroxidase; ROS, reactive oxygen species; SOD, superoxide dismutase; SA, salicylic acid; Trx, thioredoxin. ∗ Corresponding author. Tel.: +36 62 544 307; fax: +36 62 544 307. E-mail address: [email protected] (J. Csiszár). http://dx.doi.org/10.1016/j.jplph.2014.12.014 0176-1617/© 2015 Elsevier GmbH. All rights reserved.

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AtGPX4, -5 and -6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AtGPX8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cis-acting regulatory elements in AtGPX genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The role of GPXs in the development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix A. Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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The plant glutathione peroxidases are members of the thioredoxin-dependent peroxidase family Peroxidases are known to be implicated in many physiological and developmental processes, including defenses against pathogen infection, wounding and other abiotic stresses. They control cell growth either by restriction or promotion of cell elongation; they have a role in auxin catabolism, destruction of flavonoids, biosynthesis of ethylene and secondary metabolites (Welinder et al., 2002; Liszkay et al., 2003; De Gara, 2004; Passardi et al., 2004; Cosio and Dunand, 2009; Csiszár et al., 2012). Peroxidases oxidize various substrates utilizing H2 O2 or organic hydroperoxides, thus they are involved in scavenging of reactive oxygen species (ROS). ROS are natural byproduct of the normal metabolism and have important roles in cell signaling and control of redox homeostasis, while unbalanced generation of these species induces detrimental oxidation of macromolecules including DNA, proteins, and lipids. To keep the ROS level tightly regulated and to minimize ROS-derived damage, different non-enzymatic and enzymatic antioxidant systems have been evolved in aerobic organisms, including the diverse super-family of peroxidase enzymes. Peroxidases can contain a heme cofactor in their active site (such as ascorbate peroxidases or guaiacol peroxidases) or possess redox active cysteine or selenocysteine residues (non-heme peroxidases) (http://peroxibase.toulouse.inra.fr; Koua et al., 2009). The nonheme peroxidases comprise thiol peroxidases, such as thioredoxin peroxidases or peroxiredoxins (Prxs) and glutathione peroxidases (GPXs). A very conserved catalytic cysteine near the N-terminus of these proteins is called the peroxidatic cysteine (CysP -S− ) and is used to reduce hydroperoxides and peroxynitrites. This Cys residue is first transformed into a sulfenic acid (CysP -SOH) when exposed to peroxides. The main difference between the different classes is the mechanism of regeneration of the CysP -SOH, which can be reduced directly (1-Cys mechanism) or by involving a second, socalled resolving Cys residue (CysR -SH), which condenses with the sulfenic acid to form a disulfide (2-Cys catalytic cycle). The 2-Cys disulfide is reduced by thioredoxin – a low-molecular weight protein with two vicinal Cys residue – or by glutathione (reduced form GSH, ␥-glu-cys-gly) (Toppo et al., 2009). The plant thiol peroxidases can be classified into five subgroups which includes the 2-Cys Prx, 1-Cys Prx, type II Prx, Prx Q and GPX type peroxidases (Rouhier and Jacquot, 2005). In Arabidopsis thaliana 18 thiol peroxidases were identified: one 1-Cys Prx, two 2-Cys Prxs, six type II Prxs, one type Q Prx and eight GPXs (http://peroxibase.toulouse.inra.fr; Koua et al., 2009). The mammalian glutathione peroxidases (GPxs) The term glutathione peroxidase (EC 1.11.1.9.) was introduced by Mills who discovered the reaction with H2 O2 in enzyme preparations from mammalian red blood cells (Mills, 1957); the generaly accepted abbreviation of them is GPxs. They are central components of animal antioxidant metabolism and participate largely in the repair of biomembranes (Imai and Nakagawa, 2003). While GPx1–3, 5 and 6 function as homotetramers, GPx4, 7, and 8 are

monomers. The selenium-containing mammalian GPxs (GPx1–4 and, in human only, GPx6) as well as the cysteine-containing GPx-isoforms (GPx5, 7 and 8) were shown to be key players in important biological processes far beyond the detoxification of hydroperoxides (reviewed by Margis et al., 2008; Brigelius-Flohé and Maiorino, 2013). GPxs are involved in balancing the H2 O2 homeostasis in signaling cascades, e.g. in the insulin signaling pathway by GPx1 (Loh et al., 2009; Lubos et al., 2011; Brigelius-Flohé and Maiorino, 2013). GPx1 was shown to prevent oxidative DNA damage and reduce the initiation of carcinogenesis (Baliga et al., 2007; Brigelius-Flohé and Kipp, 2009). The GPx2 has anti-inflammatory function and plays a dual role in carcinogenesis depending on the mode of initiation and cancer stage (Dittrich et al., 2010; BrigeliusFlohé and Kipp, 2012). The GPx3 is membrane associated and its reduced expression or activity was also connected to many types of inflammation and cancer, even to obesity, which might be associated with oxidative stress (Lee et al., 2005, 2008; Burk et al., 2011). GPx4 enzymes can directly interfere with hydroperoxidized phospholipids in biomembranes (they are also called phospholipid hydroperoxide glutathione peroxidases, PHGPXs). Moreover, they have been reported to have a role in the regulation of apoptosis and, together with GPx5, in male fertility (Conrad et al., 2005; Seiler et al., 2008). Functions of GPx6–8 are largely unknown, although GPx7 and GPx8 were suggested to be involved in the reoxidation of protein disulfide isomerases (PDIs) during folding of proteins in the endoplasmic reticulum (Brigelius-Flohé and Maiorino, 2013). GPx7 was recently identified as an oxidative stress sensor/transducer that senses and transmits cellular ROS signals to downstream mediators to reduce ROS accumulation (Chang et al., 2013). The plant glutathione peroxidases The plant glutathione peroxidases (their more often used abbreviation is GPXs) are ubiquitous enzymes which have been shown to be present in different plant tissues, compartments and developmental stages (Mullineaux et al., 1998; Yang et al., 2005, 2006). The Arabidopsis genome contains eight GPX genes (Table 1) whose expression can be induced by multiple signals (Sugimoto and Sakamoto, 1997; Chang et al., 2009; Gaber et al., 2012; Passaia et al., 2014). An alignment of different plant GPX proteins showing conserved amino acid motifs and Cys residues is shown in Supplementary Fig. S1. In plants, the glutathione-dependent peroxidase activity can be associated with glutathione transferase (GST) isoenzymes. Their role in detoxifying lipid hydroperoxides and other reactive molecules has been shown in different species and under several stress conditions (Roxas et al., 1997; Csiszár et al., 2004; Kilili et al., 2004; Basantani and Srivastava, 2007; Dixon et al., 2009; Edwards and Dixon, 2009). Plant GPX genes with significant sequence homology to the animal phospholipid hydroperoxide glutathione peroxidases (GPx4/PHGPX enzymes) have also been isolated from several plants. All the plant GPXs characterized so far are in monomeric form (Navrot et al., 2006) except for the poplar GPX5, which showed an unique dimerization pattern mainly depending on hydrophobic contacts and was able to interact with

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Table 1 Cellular localization of the glutathione peroxidases in Arabidopsis thaliana on the basis of TAIR—The Arabidopsis Information Resource database (Garcia-Hernandez et al., 2002; Rhee et al., 2003). Name

TAIR accession number

Localization in the cell

References

AtGPX1

AT2G25080

Chloroplast

AtGPX2

AT2G31570

AtGPX3

AT2G43350

AtGPX4 AtGPX5

AT2G48150 AT3G63080

Cytosol, mitochondrion, nucleus, plasma membrane Golgi apparatus, cytoplasm, endosome, mitochondrion, trans-Golgi network Cytosol, mitochondrion Endoplasmic reticulum, plasma membrane

Rodriguez Milla et al. (2003) and Chang et al. (2009) Rodriguez Milla et al. (2003), Xu et al. (2010) and Nikolovski et al. (2012) Miao et al. (2006) and Nikolovski et al. (2012)

AtGPX6

AT4G11600

Apoplast, chloroplast, cytosol, mitochondrion, plasma membrane

AtGPX7

AT4G31870

Chloroplast

AtGPX8

AT1G63460

Cytosol, nucleus

Cd2+ ions (Koh et al., 2007). The plant GPXs contain cysteine instead of selenocysteine in their active site, which can be the reason of their lower activities when compared to their mammalian counterparts. When the selenocysteine was exchanged for cysteine by site-directed mutagenesis, the catalytic activity of the mammalian GPx decreased by two to three orders of magnitude (Maiorino et al., 1995). The plant enzymes generally use thioredoxin (Trx) as a reducing agent rather than glutathione (Navrot et al., 2006), and are regarded to be actually thioredoxin peroxidases (Herbette et al., 2002). Some of the plant GPXs were shown to have both glutathione peroxidase and thioredoxin peroxidase functions, but the thioredoxin regenerating system is much more efficient than the glutathione system (Herbette et al., 2002). Others found that plant GPXs uses only Trx but not GSH in reduction of hydroperoxides (Jung et al., 2002; Iqbal et al., 2006; Herbette et al., 2007). According to the enzymatic properties of the investigated Arabidopsis GPX isoenzymes, the KM values for Trx and H2 O2 were 2.2–4.0 and 14.0–25.4 ␮M, respectively (Iqbal et al., 2006). The catalytic efficiency (kcat /KM ) of the plant GPXs is generally around 103 to 106 M−1 s−1 , a value similar to that of various Prxs but low compared to that of ascorbate peroxidases, catalases, or human Gpxs (107 to 108 M−1 s−1 ) (Hoffmann et al., 2002; Jung et al., 2002; Herbette et al., 2005; Iqbal et al., 2006; Rouhier and Jacquot, 2005; Dietz, 2011). Plant GPXs were suggested to be more efficient in reducing peroxides different from H2 O2 such as organic hydroperoxides and lipid peroxides (Rodriguez Milla et al., 2003).

The role of plant GPXs in stress responses The plant GPXs and the oxidative stress The level of H2 O2 or alkyl hydroperoxides and other reactive oxygen species, such as superoxide radical (O2 •− ), hydroxyl radical (OH• ), singlet oxygen (1 O2 ) can be elevated either by their enhanced production or the decreased activity of the defense system. Under oxidative stress conditions the activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POD), GPX, GST and glutathione reductase (GR) are generally increased in plants and, in several cases their activities correlate well with enhanced tolerance (Foyer et al., 1997). The study of plant GPXs started with the cloning and characterization of the first cDNA from Nicotiana sylvestris, which showed high degree of similarity to animal GPxs, but without selenocysteine encoded by the terminal TGA (Criqui et al., 1992). A citrus salt-stress associated protein, which showed increased expression in NaCl-treated plants and in cultured citrus cells,

Rodriguez Milla et al. (2003) Rodriguez Milla et al. (2003) and Marmagne et al. (2004) Rodriguez Milla et al. (2003), Brugière et al. (2004), Marmagne et al. (2004), Zybailov et al. (2008) and Ito et al. (2011) Rodriguez Milla et al. (2003) and Chang et al. (2009) Gaber et al. (2012)

showed also similarity to animal glutathione peroxidases and later proved to be a PHGPX (Holland et al., 1993; Beeor-Tzahar et al., 1995; Avsian-Kretchmer et al., 1999). Early reports on plant GPXs described the identification of stress-related genes with significant sequence homology to the animal GPx4/PHGPX enzymes. Isolation and characterization of several other plant GPX coding cDNAs were reported and confirmed the role of this enzyme family in stress responses. Analysis of their gene expression showed that GPX mRNA steady-state levels usually increase under different biotic and abiotic stresses (see Reviews by Rodriguez Milla et al., 2003; Herbette et al., 2007). GPX genes were shown to be induced by oxidative stress (Li et al., 2000; Yang et al., 2005), pathogen infections (Criqui et al., 1992), mechanical stimulation (Depège et al., 1998), salt, cold, drought and metal treatments (Rodriguez Milla et al., 2003; Kang et al., 2004; Navrot et al., 2006; Diao et al., 2014; Fu, 2014; Gao et al., 2014). Transcript profiling of soybean roots showed that GPXs are among the most abundant RNAs in dehydrated tissues (Ferreira Neto et al., 2013). Detailed characterization of six Lotus japonicus GPXs revealed that GPXs have organ specific expression pattern and in response to salinity, cadmium, aluminium and nitric oxide, some of them showed up-regulation and others were down-regulated (Ramos et al., 2009). Similarly, among the three investigated Hordeum vulgare GPXs, the transcript levels of the presumably chloroplastic and cytosolic isoforms increased, whereas that of the peroxisomal ones decreased after applying different stresses (Churin et al., 1999). Passaia et al. (2013) reported that rice GPX gene family was induced in response to exogenous H2 O2 and cold treatments, while they were down-regulated after drought and UV-B light. Kim et al. (2014) demonstrated that ginseng GPXs were induced by chilling and salt stress, but showed different responses under biotic stress. Biological function of plant GPXs was extensively studied by employing transgenic plants engineered to enhance or reduce GPX pools. Transgenic tomato, in which GPX was overexpressed, showed higher tolerance towards an abiotic stress (mechanical stimulation), but lower to biotic stresses (either to a necrotrophic parasite, Botrytis cinerea, or to a biotrophic parasite Oidium neolycopersici), supposedly because higher GPX activity interfere with H2 O2 mediated signal transduction under pathogen infection (Herbette et al., 2011). These results suggest a key role for GPXs in controlling biotic and abiotic stress responses.

GPXs and the redox regulation The common feature of different environmental stresses (among others chilling, desiccation, salinity and heavy metals) is that they increase ROS production, and perturb the redox state

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of the cells (Kocsy et al., 2001; Mittova et al., 2003; Shabala, 2009; Szalai et al., 2009; Jubany-Marí et al., 2010; Miller et al., 2010). Intracellular accumulation of individual ROS and reactive nitrogen species (e.g. nitric oxide, NO; peroxinitrite, ONOO− ), ROSproducing enzymes, antioxidants and their oxidation/reduction states all contribute to the general redox homeostasis in the plant cell. Cells contain large numbers of different redox-active compounds, such as NADH and NADPH, ascorbate (ASC), glutathione, phenolics, carotenoids, cytochromes, tocopherols, polyamines and proteins carrying redox-active S-groups which are in close interaction (Foyer and Noctor, 2005; De Tullio, 2010; Potters et al., 2010). A tight control is needed to balance these activities and maintain coordination, including reversible redox regulation of proteins by dithiol–disulfide exchange, regulation of phosphoproteins, activation of signaling pathways by ROS-responsive regulatory genes (Mittler et al., 2004; Noctor et al., 2007; Foyer and Noctor, 2009; Miller et al., 2010; Dietz, 2011; Suzuki et al., 2012). Among the primary targets of ROS are amino acids such as Cys, the tripeptide GSH and protein Cys residues (D’Autréaux and Toledano, 2007; Munné-Bosch et al., 2013). ROS and particularly H2 O2 can oxidize the redox-sensitive proteins directly or indirectly via e.g. GSH or Trx. Thiol–disulfide exchange reactions, which are rapid and readily reversible, are ideally suited to control protein function via modification of the redox state of structural or catalytic SH groups (Motohashi et al., 2001; Laloi et al., 2004). Redox-sensitive proteins may modulate corresponding cellular metabolism similarly to the yeast Yap1, a transcription factor (TF) involved in oxidative stress. Yap1 contains two cysteine-rich regulatory domains that can be oxidized by ROS or thiol-active electrophiles resulting in intramolecular disulfide bond formation and nuclear localization (Delaunay et al., 2002; Herrero et al., 2008). The yeast 2-Cys GPx-type thiol peroxidase “Orp1” enzyme can sense H2 O2 by being oxidized to its sulfenic acid form. The Cys-sulfenic acid residue of Orp1 then forms a disulfide bridge with a specific thiol of the Yap1 TF (D’Autréaux and Toledano, 2007; Brigelius-Flohé and Maiorino, 2013). Delaunay et al. (2002) suggested that the ROS modification of Yap1 requires a physical interaction with yeast glutathione peroxidase 3 which functions as the primary ROS sensor. The thiol-dependent redox regulation is more diverse in plants than in animals, bacteria or fungi (Dietz et al., 2006). The occurrence of thiol-dependent activities of plant GPX isoenzymes suggests that – besides detoxification of H2 O2 and organic hydroperoxides – they can be involved in regulation of the cellular redox homeostasis by maintaining the thiol/disulfide or NADPH/NADP+ balance. Chen and his co-workers reported that tomato GPX1 (earlier LePHGPX) functioned as a cytoprotector in yeast and tobbaco, preventing Bax-, H2 O2 -, heat- and salt stress-induced cell death. Furthermore, stable expression of this GPX in tobacco conferred protection against the fungal phytopathogen Botrytis cinerea (Chen et al., 2004). Tomato plants overexpressing a mammalian selenium-independent GPx maintained a significantly higher photosynthesis rate and fructose1,6-bisphosphatase activity under chilling stress, and the role of modified levels of antioxidant ASC and GSH – the main redox couples – was suggested in the sustained viability (Herbette et al., 2005). The relationship of GPX and redox regulation was demonstrated recently in the reprogramming of the ROS gene expression network in acclimation of Brassica rapa var. nipposinica (Mizuna) plants to long-term spaceflight environment. The transcription of a glutathione peroxidase was up-regulated more than two-fold together with other major ROS-scavenging enzymes as superoxide dismutase (SOD) and catalase, in parallel more than 40-fold induction occurred in thioredoxin and glutaredoxin (GRX) genes, which are crucial for maintaining the reduced intracellular redox state and oxidative defense in the distinct conditions (Sugimoto et al., 2014).

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GPXs interact with other proteins and hence they are considered to have signaling functions (Delaunay et al., 2002) moreover, they were suggested to function as a ROS- or redox sensors (Rodriguez Milla et al., 2003). According to Miao et al. (2006) the redox state of the Arabidopsis AtGPX3 is regulated by H2 O2 and this GPX functions as a redox transducer in abscisic acid (ABA) and drought stress signaling. The AtGPX3 interacts with protein phosphatase type 2C (PP2C) proteins such as abscisic acid insensitive (ABI) 1 and ABI2, in order to activate plasma membrane Ca2+ and K+ channels that facilitate stomatal closure (Miao et al., 2006), (more details can be found below). Overexpression of two putative Triticum aestivum GPX genes in Arabidopsis conferred high tolerance to salt, H2 O2 and ABA treatments. The altered expression levels of ABI1, ABI2, SOS1 and RbohD genes (key regulators involved in salt, H2 O2 and ABA signaling) in the transgenic Arabidopsis lines indicated also the role of GPXs in salt- and ABA signaling (Zhai et al., 2013). Arabidopsis thaliana glutathione peroxidases—Lessons from a model plant In Arabidopsis thaliana 8 members of plant glutathione peroxidases have been identified. The Arabidopsis GPX proteins are monomeric, their size range between 167 and 236 amino acids (Supplementary Fig. S1). Although the core of the proteins is well conserved, the N-terminus is highly variable, mostly because of the presence of transit peptides which were identified in this region of some genes. Phylogenetic analysis of Arabidopsis GPXs revealed that three pairs of proteins show stronger similarity to each other than to the rest of the family (Rodriguez Milla et al., 2003; Chang et al., 2009). Extended phylogenetic analysis of AtGPXs with known GPXs from other Brassicaceae revealed that beside the AtGPX1 and AtGPX7 (chloroplastic group), AtGPX4 and AtGPX5, AtGPX6 and AtGPX8 pairs, the AtGPX2 and AtGPX3 are also in a close relationship (Supplementary Fig. S2), presumably as a consequence of whole genome duplication events in the ancestral Angiosperms (Bowers et al., 2003). Some of them are abundant and distributed in most of the cell organelles (e.g. AtGPX2, AtGPX3, AtGPX6), while the occurence of others is restricted to one or two subcellular compartments (Table 1). AtGPX1, -2, -5 and -6 were overexpressed in Escherichia coli and characterized by Iqbal et al. (2006). The recombinant proteins were able to reduce H2 O2 , cumene hydroperoxide, phosphatidylcholine and linoleic acid hydroperoxides using Trx but not GSH or NADPH as an electron donor. The reduction activities of the recombinant proteins with H2 O2 were 2–7 times higher than those with cumene hydroperoxide. Roles of the different AtGPXs were recently investigated using T-DNA insertion mutants by Passaia et al. (2014). The phenotypes were largely similar to wild type in all mutant lines, suggesting that these isoenzymes are substantially redundant or other components of the antioxidant system can compensate the loss of a particular GPX. However, they also demonstrated discrete roles of individual isoenzymes, because some differences in the number of rosette leaves and lateral roots of the 4-week-old plants were observed (Passaia et al., 2014). AtGPX1 and AtGPX7—The chloroplastic isoenzymes The AtGPX1 and AtGPX7 have 82% amino acid identity and are considered to be specific for chloroplasts. On the basis of high homology it was supposed that they likely have overlapping activities (Rodriguez Milla et al., 2003), however their responses to different stimuli differ considerably based on meta-analysis of gene expression data (Chang et al., 2009). It was reported that the chloroplastic GPXs (cpGPXs) contribute to cross-talk between photooxidative stress and immune responses (Chang et al., 2009). AtGPX1 but not AtGPX7 might be involved in defence against

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virulent pathogen infection. Reduced cpGPX expression either in antisense (AS) transgenic lines with lower total cpGPX expression (GPX1 and GPX7) or in Atgpx7 insertion mutant resulted in higher foliar H2 O2 levels in excess light, reduced photooxidative stress tolerance but increased basal resistance to virulent bacteria. The AS-cpGPX lines displayed enhanced cell death and resistance to Pseudomonas bacteria compared with the wild type. Constitutively reduced cpGPX expression caused decreased Cu/ZnSOD and MnSOD activities, higher foliar salicylic acid (SA), ASC, GSH and other antioxidant levels in plants exposed to higher light intensities. Interestingly, no significant differences were observed in lipid peroxidation even under excess light. Additionally, alterations were found in leaf mesophyll and chloroplast morphology. Leaf tissues were characterized by shorter and more rounded palisade cells, irregular spongy mesophyll cells, and larger intercellular air spaces compared with the wild type. Chloroplasts had larger and more abundant starch grains in AS-cpGPX transgenic lines than in wild type and Atgpx7 mutant plants. These results indicate the involvement of cpGPXs in light acclimation, leaf development and cell death programmes. Their important role was suggested in maintaining chloroplastic ROS homeostasis and redox signaling between cellular compartments that may coordinate acclimatory and defense responses (Chang et al., 2009). Passaia et al. (2014) demonstrated that AtGPX7 but not AtGPX1 have a role in the pathways that control shoot growth. According to microarray data found in Genevestigator database (https://www.genevestigator.com/gv/plant.jsp; Hruz et al., 2008), the expression levels of AtGPX1 and AtGPX7 are high in shoot, sepal and petal, but are low in the root and fascicular tissues (Supplementary Fig. S3). Their expression was induced commonly by high light or transferring etiolated plants to light. In addition, the AtGPX7 transcription was induced by UV-B, blue light, gibberellic acid (GA), indoleacetic acid (IAA), methyl jasmonate (MeJA), cold, drought and during growth of pollen tube (Supplementary Fig. S4). The transcript level of AtGPX1 decreased during germination and callus formation, under cold, osmotic and drought stress. Different virulent and avirulent biotrophic pathogens (Pseudomonas syringae pv. tomato/phaseolicola/maculicola, Golovinomyces orontii) and elicitors also down-regulated this gene, of which encoded protein may limit programmed cell death in response to infection (Chang et al., 2009). The expression of AtGPX7 was downregulated by hypoxia, short-day, germination and in the presence of several fungi (Hyaloperonospora arabidopsidis, Golovinomyces cichoracearum, Pseudomonas syringae pv. tomato) (Supplementary Fig. S4). AtGPX2 The AtGPX2 is a poorly investigated isoenzyme which was found in several tissues (Supplementary Fig. S3) and localized in several cell compartments (Table 1). Xu et al. (2010) showed that AtGPX2 can be part of a cytosolic protein complex involved in stress defense. In vitro and in vivo studies proved that AtDJ-1a (a homolog of animal DJ-1 superfamily protein, also known as PARK7, Parkinson protein 7) similarly to the DJ-1/PARK7 act as adaptor proteins that bring together SOD and AtGPX2 in a conformation that allows damaging O2 •− to undergo SOD-mediated dismutation into O2 and H2 O2 . The supposed role of AtGPX2 is to convert H2 O2 into H2 O (Xu et al., 2010). Public transcript profiling data showed that the AtGPX2 transcript is abundant in all developmental stages, in particular in protoplasts and shoot cell culture (Genevestigator, Supplementary Fig. S3). In 10-day-old seedlings the level of AtGPX2 mRNAs was the highest in the root and it showed an equally high value also in the shoot (Passaia et al., 2014). The expression of AtGPX2 gene notably increased during germination, pollen tube growth and

shoot regeneration, furthermore it was activated by ABA and SA treatments, drought, osmotic stress and some fungi (Supplementary Fig. S4). These responses suggest that AtGPX2 can be implicated in stress response and development. However, the gene was downregulated by MeJA and brassinolide treatments, during formation of callus and on long-day conditions. The level of transcription is low in pollen and fascicular tissues (Supplementary Fig. S3). AtGPX3 The A. thaliana GPX3 proved to be a monomeric transmembrane protein which is located in the Golgi apparatus, cytoplasm, endosome, mitochondrion and trans-Golgi network (Table 1). It has outside membrane regions comprising 170 amino acids (Li et al., 2013b). Investigation of Atgpx3 insertion mutant plants revealed that these mutants are sensitive to and produce more H2 O2 than wild type (Miao et al., 2006). It was suggested that AtGPX3 plays a dual role in stress responses: on one hand it contributes to the maintenance of H2 O2 homeostasis by the elimination of H2 O2 and organic hydroperoxides, on the other hand, AtGPX3 works as a switch in ABA and H2 O2 signaling pathway in guard cell, thereby controling the transpiration. Mutation of the AtGPX3 disrupted ABA activation of calcium channels and the expression of ABA and stress-responsive genes. Bimolecular fluorescence complementation (BiFC) assays revealed, that AtGPX3 physically interacts with the 2C-type protein phosphatase ABI2 and to a lesser extent with ABI1, which are known to be important players in ABA signaling. Compared with wild type plants, Atgpx3 mutants displayed impaired ABA and H2 O2 -induced stomatal closure, faster water loss, and lower leaf temperatures in response to water deficit stress (Miao et al., 2006). Atgpx3 plants proved to be more sensitive to drought and osmotic stress than the wild type (Miao et al., 2007). In AtGPX3 overexpressing lines the water loss of detached leaves was significantly lower, the leaf surface temperature was higher compared to wild type plants (Miao et al., 2006). Interestingly, results related to the growth of Atgpx3 roots under ABA treatment suggested no specific roles of AtGPX3 in the ABA-dependent control of root architecture (Passaia et al., 2014). According to public transcript profiling data (Genevestigator database) transcription of AtGPX3 is activated under germination, formation of callus, hypoxia, cold and salicylic acid treatment, furthermore it is high in shoot apex, replum, primary root, abscission zone and root apical meristem. On the other hand, its expression level is low in protoplast, pollen, phloem, endosperm, and decreased under zeatin treatment, preparation of protoplast and when etiolated plants are transferred to light (Supplementary Fig. S4). AtGPX4, -5 and -6 The first Arabidopsis cDNA encoding a putative PHGPX was cloned and sequenced by Sugimoto and Sakamoto (1997), which now is designated as the AtGPX6. The expression profile of this gene under NaCl-, Al- and Fe treatments – which triggers peroxidation of membrane lipids – suggested that it is induced by oxidative stress (Sugimoto and Sakamoto, 1997; Noctor et al., 2011). Despite of this, the AtGPX6 together with AtGPX4 and AtGPX5 belongs to the poorly investigated GPXs of Arabidopsis. While a comprehensive phylogenetic analysis of Margis et al. (2008) implied that AtGPX4 and AtGPX5 are cytosolic isoenzymes and the AtGPX6 is localized to the mitochondrion, there is some controversy regarding their intracellular occurrence (Rodriguez Milla et al., 2003; Passaia et al., 2014; Table 1). Biochemical characterization of recombinant proteins revealed that both AtGPX5 and AtGPX6 can detoxify H2 O2 and organic hydroperoxides using thioredoxin in vivo, although AtGPX5 was not able to reduce cumene hydroperoxide with either

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Trx or GSH. Beside the different role of isoenzymes, these results suggested that GPXs may work in tandem with peroxiredoxins, the other antioxidant enzymes utilizing Trx, to detoxify H2 O2 and organic hydroperoxides and also be involved in the regulation of the redox homeostasis by maintaining the thiol/disulfide or NADPH/NADP+ balance (Iqbal et al., 2006). Gene expression data show that the expression of AtGPX4 is notable in pollen, stamen and phloem, but very low in other organs or tissues and during germination, growth of pollen tube and repressed further by brassinolide treatment (Supplementary Figs. S3 and S4). The AtGPX4 mRNA was below the level of detection in roots and shoots of the 4-week-old seedlings (Passaia et al., 2014). The transcription level of AtGPX5, similarly to the AtGPX4, is high in pollen and increased during growth of pollen tube (Supplementary Fig. S4). In addition, it is induced by drought, SA and zeatin treatments, and is upregulated in the presence of different fungi (H. arabidopsidis, G. cichoracearum, P. syringae pv. maculicola). The expression of the gene decreased under cold and heat stress, high light and during formation of callus, and it is low in the interfascicular cambium and stigma. The level of transcription of AtGPX6 is increased by most of abiotic stresses which correlates to its role in abiotic stress response (Rodriguez Milla et al., 2003). It is induced during cold, salt stress, high light, sucrose and PEG treatments, after applying ABA, MeJA, or zeatin and during the growth of pollen tube. Its expression is high in protoplasts and guard cells, but decreased during formation of callus and germination (Supplementary Figs. S3 and S4). AtGPX8 Gaber et al. (2012) reported that AtGPX8 is localized in the cytosol and nucleus, although the majority of AtGPX8 was in the cytosol. AtGPX8 is supposed to play an important scavenging role and prevents damages of DNA under oxidative stress. In fact, the overexpression and disruption of AtGPX8 conferred increased and decreased tolerance to oxidative stress, respectively. Localization of AtGPX8 in the nucleus indicates that it is involved not only in the protection of cellular components but also in redox modification of nuclear proteins with putative signaling functions. It was suggested that ubiquitin/proteasome dependent proteolysis is involved in the rapid turnover of AtGPX8 protein under stress (Gaber et al., 2012). The AtGPX8 transcript level increased not only on account of abiotic stress but even of different hormones (indole-3-acetic acid, ABA, SA, jasmonic acid), indicating that these hormones are associated with the regulation of AtGPX8 gene expression (Gaber, 2011). According to the data found in Genevestigator, the expression of AtGPX8 is increased by hypoxia, germination, formation of callus, short term ABA and SA treatments and long term cold, but during long term ABA treatment and drought its expression decreased. The level of transcription is the highest in the tissues of root, but in the pollen and seed is very low (Supplementary Fig. S3). Cis-acting regulatory elements in AtGPX genes While information about biochemical features of AtGPX proteins and their protein-protein interactions is limited, analysis of promoter regions of their respective genes can help characterize regulation of genes and their regulatory networks. Several AtGPX genes were upregulated in response to abiotic stresses or hormone treatments (Rodriguez Milla et al., 2003). Actual information by in silico screening of cis-regulator elements present in the ca. 1 kb of the 5 regulatory regions from the translational start sites of the AtGPX genes using PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html) are summarized in Supplementary Table S1.

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Multiple putative cis-acting regulatory elements with different number were detected in the upstream regions of each AtGPX genes (Rodriguez Milla et al., 2003; Gao et al., 2014). The presence of different abiotic and biotic stress related cis elements in the promoters of glutathione peroxidase genes supports their involvement in abiotic and biotic stress responses. Moreover, comparison of the Arabidopsis and the more stress tolerant relative Thellungiella salsuginea revealed higher number of stress- and hormone response related cis-acting regulatory elements in the promoters of TsGPXs than AtGPXs (Gao et al., 2014). In Arabidopsis, the drought responsive MYB binding site was found in the 5’ regulatory regions of AtGPX2, -3, -5, -6, -7 and -8, the stress related TC-rich repeats in the promoters of AtGPX1, -2, -6, -7 and -8 genes. HSE element, involved in heat stress responsiveness, was present in 5 regions of AtGPX1, -2, -5, -6 genes. Elicitor responsive elements (EIRE, BoxW1) were identified in the 5 regulatory region of AtGPX1, -2, -4, -5 and wound-responsive element in AtGPX7 genes. ARE regulatory element, essential for the anaerobic induction, was found in the promoter of AtGPX4 and -5. Furthermore, all but the Arabidopsis GPX3 gene contained a high number of different kind of light responsive elements (ACE, AE-box, AT1- motif, ATC-motif, Box 4, Box I, Box II, CATT-motif, G-box, GA-motif, GAG-motif, Gap-box, GATA-motif, GT1-motif, I-box, TCCC-motif, TCT-motif, as-2-box or Chs-CMA1a) and regulatory element involved in circadian control was predicted at AtGPX1, -3, -5 and -8 (Supplementary Table S1). Among the cis-acting elements involved in different hormone regulation abscisic acid responsive elements (ABRE, CE1) were found in AtGPX1, -2 and -6, MeJA and auxin response elements in AtGPX2, -6, -7 and -8. Ethylene responsive element (ERE) was present in the upstream regulatory region of AtGPX1, -4 and -6, whereas gibberellin responsive elements (GARE, P-box) in AtGPX6 and -8. Interestingly, meristem specific cis-regulatory elements (CATbox and dOCT) in AtGPX5, -6, -8, and seed development specific sequences (AAGAA-motif, ATGCAAAT motif, GCN4 motif, O2-site, Skn-1 motif, TATCCAT/C-motif) were also identified in most of AtGPX genes except for AtGPX4 (Supplementary Table S1). Although the proposed function of these motifs should be considered with caution, their presence suggests that expression of the GPX genes is under complex transcriptional control.

The role of GPXs in the development Several reports have demonstrated that plant hormones (e.g. auxin, GA, ABA, SA, MeJA) can regulate the expression of GPX genes in different species (Li et al., 2013a; Zhai et al., 2013). Public microarray data of AtGPXs expression show tissue- and developmental-dependent differences in their transcript levels indicating their involvement in the control of plant development. For example, during the germination some genes are activated (AtGPX2, -3 and -8), while others (AtGPX1, -4, -6 and -7) are repressed (Supplementary Fig. S4). According to their relatively high expression levels, the AtGPX2, -3, -5, -6 and -8 gene products may play a role in the growth and differentiation of roots (Fig. 1, Supplementary Fig. S3). The extremly high expression level of AtGPX1, -2 and -6 in leaf cell- and mesophyll protoplast cultures, furthermore the relatively high transcription of the AtGPX1, -2, -3 and -6 in seedlings, rosette leaves and especially in shoot apical meristems suggest the physiological importance of the encoded isoenzymes in shoot development. The role of AtGPX4 and -5 enzymes in pollen tube growth was indicated on the basis of the very high gene expression levels found in flowers, especially in stamen and pollen (Supplementary Figs. S3 and S4). The AtGPX5 was also demonstrated as a female gametophytic development related gene by performing a large-scale insertional mutagenesis screen using an Ac/Ds transposon system, because the endosperm

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Fig. 1. The expression levels of the eight Arabidopsis thaliana GPX genes at different stage of development, using Genevestigator (https://www.genevestigator. com/gv/plant.jsp) programme’s Development tool.

development was arrested in the mutants (Pagnussat et al., 2005). One Avena fatua GPX was even identified as nondormancy associated gene (Johnson et al., 1995). These data implicate that GPXs play a complex role in plants during their whole life, from germination to seed development, furthermore, they may even regulate the development. Although precise data on their function in developmental regulation is scarce, use of transgenic plants and mutants have recently shed some light on their role in plant development. Silencing of the rice mitochondrial OsGPX3 gene resulted in impaired normal plant development, as these plants had shorter roots and smaller shoots comparing to non-transformed plants, and higher amounts of H2 O2 were released from the root mitochondria under normal conditions (Passaia et al., 2013). Passaia et al. observed some differences in the number of rosette leaves of the 4-week-old T-DNA insertion mutants of Arabidopsis GPXs grown under short day conditions comparing to the wild type (Passaia et al., 2014). Moreover, Atgpx1, Atgpx4, Atgpx6, Atgpx7 and Atgpx8 mutants had a significantly greater lateral root density than the wild type, while that of the Atgpx2 and Atgpx3 mutants were significantly lower. The role of GPXs in the hormone-mediated (auxin, strigolactone, ABA) control of lateral root development was also demonstrated (Passaia et al., 2014). Furthermore, GPXs may affect plant regeneration. Faltin et al. (2010) tried to obtain constitutive overexpression of citrus (Citrus sinensis) cit-PHGPx in transgenic plants. All attempts to obtain regenerated plants expressing this enzyme constitutively failed. However, when the enzyme’s catalytic activity was abolished by site-directed mutagenesis of the catalytic residue (Cys41) into

serine (Ser41), transgenic plants constitutively expressing inactive cit-PHGPx were successfully regenerated. Constitutive expression of enzymatically active cit-PHGPx could only be obtained when transformation was based on non-regenerative processes. These results indicate that overexpression of the GPX interferes with shoot organogenesis. GPX most probably modulate ROSdependent developmental and metabolic signals, which can alter differentiation-related events during plant regeneration. Monitoring the ROS level during regeneration revealed that upon cit-PHGPx induction, the lowest level of ROS coincided with the maximal level of shoot inhibition. Correlation was found between the reduction in ROS level and the inhibitory effect of cit-PHGPx at the early stages of in vitro shoot organogenesis (Faltin et al., 2010). However, we can suppose that GPX also affects regeneration by modulating the activities of redox sensitive thiol proteins, particularly those involved in signal transduction pathways (similarly to the microbial oxyR, Kim et al., 2002). Redox balance was shown to be critical for cell cycle progression and cell differentiation. G1-S transition of the cell cycle was shown to be a redox sensitive phase (den Boer and Murray, 2000). In case of altered redox balance, cells may stop proliferating and switch to differentiation and programmed cell death. Importance of redox balance of the glutathione pool on growth and differentiation or the root apical meristem was demonstrated (SánchezFernández et al., 1997; Vernoux et al., 2000; Kocsy et al., 2013). Glutathione is one of the most important thiol antioxidants and redox buffers and its level and redox status are key regulators of plant development and responses to the environment (see review by Noctor et al., 2011). Depending on the total concentration of

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GSH in a cell, the magnitude of an oxidative event associated with the initiation of differentiation or cell death would vary (Schafer and Buettner, 2001). The proliferating state was supposed to be connected with the calculated glutathione half-cell redox potential in plants ranging −260 mV to −210 mV, while differentiation was associated with a more positive potential, −210 to −180 mV. Above −180 mV apoptosis is triggered, and if this rises further, then necrosis will be the dominant form of cell death (Schafer and Buettner, 2001). Although the exact glutathione redox potential values of plant cells proved to be lower by using redox active fluorescent probes (Meyer et al., 2007), the correlation between the redox status and redox active compounds exists (Jubany-Marí et al., 2010). While the NAD+ , NADP+ , glutathione and ascorbate are considered to be the main redox couples (Foyer and Noctor, 2011), recent studies have suggested some redundancy between glutathione and other redox systems such as Trx (Noctor et al., 2011). Glutathione peroxidases were suggested to represent a putative link between the glutathione-based and the thioredoxin system (Jung et al., 2002; Rodriguez Milla et al., 2003). The antiapoptotic activity of GPX was also demonstrated for mammalian and plant GPXs (Chen et al., 2004; Nakagawa, 2004). During differentiation, GPXs might suppress programmed cell death, which is an integral part of many developmental processes. Alternatively, GPXs may physically interact with other proteins and hence they may have certain signaling functions (Delaunay et al., 2002; Miao et al., 2006). More speculatively, alternative substrates of GPXs such as organic hydroperoxides, or their catalytic products such as lipid derivatives, may have also some role in developmental regulation. Additional studies are thus required to clarify the respective developmental functions of GPX’s and their functional redundancy.

Concluding remarks The plant GPX family shares significant sequence homology to the animal GPx4/PHGPX enzymes, however they contain Cys residues in their active site instead of selenocystein. They were suggested to be more efficient in reducing peroxides different from H2 O2 such as organic hydroperoxides and lipid peroxides. The exact mechanisms of plant GPXs are not yet known, but they can be regarded as more than simple antioxidant enzymes. Most of plant GPXs prefer to use Trx instead of glutathione as an in vitro electron donor, GPXs may thus work in tandem with peroxiredoxins to detoxify peroxides and also be involved in the regulation of the redox homeostasis by maintaining the thiol/disulfide or NADPH/NADP+ balance. The different expression pattern and intracellular locations of plant GPXs indicate that individual isoforms have particular functions. It is still required detailed functional analysis, characterization for individual GPX isoenzymes and to identify partners that GPXs interact specifically. In stress responses, they can play multiple role: (i) maintenance of H2 O2 homeostasis by the elimination of H2 O2 and organic hydroperoxides, (ii) participation in protein complexes involved in stress defense (as e.g. AtDJ-1a, AtGPX2 and SOD), (iii) redox modification of nuclear proteins with putative signaling functions (as it was supposed for AtGPX8), (iv) contribution to cross talk between different signaling pathways, as it was suggested for AtGPX1 and AtGPX7 (photooxidative stress and pathogen infection) or AtGPX3 (H2 O2 and ABA signaling). Recent evidence suggests that GPXs not only can protect cells from stress-induced oxidative damages but they function as a crucial component of plant development and growth. They might be implicated in: (i) hormone-mediated growth of roots and lateral roots, (ii) plant regeneration and shoot organogenesis, (iii) development of leaves, (iv) flower- and seed development, (v) supressing programmed cell death (Fig. 2). However, there are several unresolved questions regarding their mechanism of action and how

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Development Hormones

Photosynthesis Respiration

Abiotic stress

Other biochemical processes

Biotic stress

Redox homeostasis NAD+/NADH, NADP+/NADPH, ROS (H2O2, ROOH), RNS Antioxidants GSH/GSSG, ASC/DHA Thiol -SH/SS, Trx, GRX

Hormone-induced signaling

?

GSH-independent ROS signaling

?

?

Thiol-disulfide signaling

Signal strength, location, specificity

GPXs Defence

Development - Hormone mediated root growth - Shoot growth and organogenesis - Regeneration - Flower- and seed development - Supressing cell death

-Crosstalk between different signaling pathways - Modification of proteins with signaling function - Elimination of ROOH, H2O2, - Involvement in cell death -? Involvement in protein complexes to promote antioxidant defence

Fig. 2. Hypothetical scheme showing links of GPXs to main physiological and biochemical processes and major signaling pathways. ROS, reactive oxygen species; RNS, reactive nitrogen species, GSH, reduced glutathione, GSSG, oxidized glutathione; ASC, ascorbic acid; DHA, dehydroascorbic acid; Trx, thioredoxin; GRX, glutaredoxin; GPXs, glutathione peroxidases.

these enzymes are involved in interactions with other proteins and how they influence developmental and stress signaling. Acknowledgements This work was supported by the Hungarian National Scientific Research Foundation [grant number OTKA K 105956] and by the European Union and the Sate of Hungary, co-financed by the European Social Fund in the framework of TÁMOP 4.2.4.A/2-11/12012-0001 ‘National Excellence Program’ scholarship to E.H. and K.B. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jplph.2014. 12.014. References Avsian-Kretchmer O, Eshdat Y, Gueta-Dahan Y, Ben-Hayyim G. Regulation of stressinduced phospholipid hydroperoxide glutathione peroxidase expression in citrus. Planta 1999;209:469–77. Baliga MS, Wang H, Zhuo P, Schwartz JL, Diamond AM. Selenium and GPx-1 overexpression protect mammalian cells against UV-induced DNA damage. Biol Trace Elem Res 2007;115:227–42. Basantani M, Srivastava A. Plant glutathione transferases—a decade falls short. Can J Bot 2007;85:443–56. Beeor-Tzahar T, Ben-Hayyim G, Holland D, Faltin Z, Eshdat Y. A stress-associated citrus protein is a distinct plant phospholipid hydroperoxide glutathione peroxidase. FEBS Lett 1995;366:151–5.

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